Hedgehog-related prophylaxis, therapy and diagnosis of gi tract carcinogenesis

ABSTRACT

The present invention is based on the key roles played by Hedgehog proteins in the regulation of homeostasis of the adult intestinal epithelium. Ihh is expressed in the adult mammalian colon and provides a lineage-instructive signal and regulates colonic epithelial morphogenesis in a compartmental fashion. Loss of Ihh expression precedes morphological change in colon tumorigenesis, i.e. carcinogenesis, and Ihh was absent in HT-29 colon carcinoma cells. Treatment of cancerous HT-29 cells with exogenous Hedgehog protein restored their differentiation. Ihh thus plays a pivotal role in the maintenance of colonic epithelial homeostasis in the differentiation of the GI tract cells and is essential for the enrolment of these GI tract cells on the Death program thus maintaining homeostasis to avoid or treat carcinogenesis. In addition, in gastric cancer expression of Shh is lost and loss of Shh expression precedes morphological changes in the parietal cells of the stomach. Shh is specifically expressed in fundic glands as well as in gastric heterotopia in the esophagus in Meckel&#39;s diverticulum. Shh thus has a unique role as a morphogen in fundic gland homeostasis. The present invention relates to methods in which a source of Hedgehog proteins is used prophylactically or therapeutically to maintain homeostasis of the adult intestinal epithelium. In particular the invention relates methods whereby sources of Hedgehog protein are used to prevent or treat carcinogenesis in adult gastric and colonic tissues. The invention also relates to Hedgehog-based method of diagnosing susceptibility for or the presence of carcinogenesis in the adult GI tract, particularly in gastric and colonic tissues. The invention further provide for compositions to be used in the Hedgehog-based methods of diagnosing, preventing and treating epithelial tumorigenesis in the adult GI tract.

FIELD OF THE INVENTION

The present invention resides in the fields of recombinant genetics, andmedicine and is directed to the use of Indian and Sonic Hedgehogproteins and nucleic acids encoding the Indian and Sonic Hedgehogproteins in the prophylaxis, therapy and diagnosis of GI tractcarcinogenesis, e.g. in gastric or colonic cancer.

BACKGROUND OF THE INVENTION

During organogenesis the cells of the endodermal layer give rise to theliver, pancreas and epithelial cells of the lung and gastrointestinal(GI) tract. The differentiation of these different organs with theirrespective functional cell types occurs through complexmesenchymal-endodermal interaction. In this interaction, the Hedgehog(hh), Fibroblast Growth Factor (Fgf), Wnt and Transforming Growth Factor(TGF)-β families of secreted proteins play a key role (Hogan, 1999).

Hedgehog was initially identified in a genetic screen for segmentpolarity genes in Drosophila (Nüsslein-Volhard and Wieschaus, 1980). Invertebrates three hedgehog genes have been identified, Sonic Hedgehog(Shh), Indian Hedgehog (Ihh) and Desert Hedgehog. All three Hedgehog'sbind to the same receptor, Patched (Ptc) which controls the activity ofa second receptor, Smoothened (Smo) (Kalderon, 2000). Both Shh and Ihhplay a role in endodermal/ectodermal-mesodermal interactions in the gut(Bitgood and MacMahon 1995; Roberts et al., 1995; Roberts et al., 1998;Litingung et al., 1998; Sukegawa et al., 2000; Ramalho-Santos et al.,2000; Kim et al., 1998; Hebrok et al., 1998; Apelqvist et al, 1997;Marigo et al. 1995)

Expression of Shh in the gastrointestinal tract has been describedduring development in many vertebrate systems including mouse (Bitgoodet al., 1995), chick (Ronberts et al. 1995), human (Ryan et al., 1998),and frog. In all species examined, Shh is expressed from the earliesttime points of gastrointestinal development restricted in its expressionto the endoderm. The murine gut has been well examined for Shh mRNAexpression throughout development. At a late stage of development, 18.5days post coitum (d.p.c.), one day prior to birth, Shh mRNA is detectedin the glandular epithelium of the stomach, the small intestine and thecolon (Apelqvist et al., 50). Thus, Shh is expressed at a late stage ofintra-uterine development, whereas at the same time the murine GI tractundergoes major morphological and functional changes during the firstthree postnatal weeks, including formation of intestinal crypts andmaturation of the gastric glands (Gordon and Hermiston, 1994; Karam etal, 1997). No information is available in the art, however, as to whathappens to Shh mRNA expression in the adult.

Several studies have addressed the functional role of Shh expression inthe developing gut. Studies in chick and mouse using either overexpression or inactivation of Shh suggest that during development Shh isa critical endodermal signal in the epithelial/mesodermal signallinginvolved in specification of differentiation along theanterior-posterior as well as the radial axis of the vertebrate gut(Bitgood and MacMahon 1995; Roberts et al., 1995; Roberts et al., 1998;Litingung et al., 1998; Sukegawa et al., 2000; Ramalho-Santos et al.,2000; Kim et al., 1998; Hebrok et al., 1998; Apelqvist et al, 1997;Marigo et al. 1995). Shh null mice display gastrointestinalmalformations including a failure of the trachea and esophagus toseparate normally, gut malrotation, small intestinal and anus atresias.The gastric epithelium of Shh null mice shows epithelial hyperplasia andalkaline phosphatase expression, a sign of intestinal differentiation(Apelqvist et al., 1997).

Despite this knowledge of the embryonic and not yet weaned mice, thereis a lack of information about Hedgehog expression in the adult andtheir role in this rapidly regenerating system. Van den Brink et al.(2001) previously showed that Shh is expressed in the fundic gland ofthe adult human and rodent stomach. Inhibition of Shh led to enhancedepithelial proliferation and diminished protein levels of BMP-4,Islet-1, and Hepatocyte Nuclear Factor 3β, all of which are proteinsinvolved in differentiation and tissue specific gene expression.Although this may indicate some role for Shh in the regulation of fundicgland homeostasis in the adult proximal stomach, there is, however, noinsight into the lineage-instructive mechanisms that regulatedifferentiation of intestinal epithelial precursor cell descendants.

After the establishment of differentiation of the GI tract along all itsaxes of development, continuous renewal of GI epithelial cells in theadult occurs along a single vertical (or radial) axis. For instance, acommon progenitor cell in the crypts of the intestine can give rise to avariety of epithelial cell types with digestive, absorptive, protectiveand endocrine functions (Stappenbeck et al., 1998; Montgomery et al.,1999; Roberts 2000). The cells differentiate as they move towards theintestinal lumen and undergo a death program thus maintaininghomeostasis (Hall et al., 1994). Although loss of this tightly regulatedepithelial differentiation is a central aspect of the development ofcolon cancer, the factors that regulate epithelial differentiation stillremain to be identified. Differentiation of the epithelial cells is acell non-autonomous process that seems to be critically dependent onpositional information along the vertical axis of renewal (Sweetser etal., 1988; Hermiston et al., 1996). Several tissue and celllineage-specific transcription factors have been identified thatregulate the expression of cell type specific markers of differentiation(Montgomery et al., 1999). However, the molecular mechanisms that timeand direct the induction of these transcription factors at theappropriate position along the vertical axis remained unresolved thusfar.

In the adult vertebrate colon, precursor cells at the bottom of thecolonic crypt can differentiate in to three cell types: absorptivecolumnar cells, goblet cells and endocrine cells (Chang et al., 1971).The endocrine cell is present in highest numbers at the base of thecrypt whereas most of the goblet cells are located in the mid-crypt. Thepredominant cell type, the absorptive enterocyte, shows a graded patternfrom a relatively undifferentiated phenotype in the crypt to a fullydifferentiated phenotype at the inter-crypt table (Chang et al., 1971).During embryogenesis cells receive the positional information thatdetermines their developmental fate from their relation to gradients ofsecreted morphogens (Hogan. 1999).

During development the GI tract is patterned throughendodermal-mesenchymal interactions. In this interplay Sonic Hedgehog(Shh) and Indian Hedgehog (Ihh) are critical endodermally derivedmorphogens (Roberts et al. 1995; Apelqvist et al., 1997; Roberts et al.,1998; Litingung et al., 1998; Ramalho-Santos et al., 2000; Mo et al.,2001; Sukegawa et al. 2000; Zhang et al. 2001). Both have partiallyoverlapping functions and act through the same complex of an Hedgehogbinding receptor Patched (Ptc), a signalling receptor Smoothened (Smo)and the Gli family of transcriptional effectors (McMahon 2000). Van denBrink et al. (2001) previously showed that Shh is involved in theregulation of fundic gland homeostasis in the adult proximal stomach.Hedgehog signalling plays an important role in the development of thehindgut and this role is conserved from fly to mice (Hoch and Pancratz1996; Takashima and Murakami 2001). Ihh mRNA is produced in the colonuntil at least one day prior to birth in mice (Ramalho-Santos et al.,2000). However, the prior art does not disclose anything on theexpression of Ihh mRNA or protein in the adult colon.

Thus, there is still a need for identification of the key molecule(s)that regulate GI tract epithelial homeostasis. As a consequence there isstill a need for compositions and methods based on such key molecule(s),that may be used in the prophylaxis, therapy and/or diagnosis of GItract carcinogenesis, such as e.g. in gastric or colonic cancer.

DESCRIPTION OF THE INVENTION DEFINITIONS

The term “Hedgehog” or “Hedgehog protein” is herein understood to mean apolypeptide with the amino acid sequence that is substantially similarto the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ IDNO: 3 (Desert, Indian and Sonic). The term “Hedgehog” is thusinterchangeably used for the human Desert, Indian and Sonic Hedgehogproteins (referred to as Dhh, Ihh and Shh, respectively) as well as fortheir non-human mammalian homologues and includes allelic forms andmuteins of these polypeptides comprising one or more amino acidsubstitutions, deletions and/or insertions. The term “Hedgehog” as usedherein thus comprises polypeptides preferably having at least 63% aminoacid identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2and SEQ ID NO: 3, preferably having at least 75, 80, 90, 95 or 99% %amino acid identity with the amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 2 and SEQ ID NO: 3.

Included in the term “Hedgehog proteins” are also proteins of the sameor similar sequence as a native Hedgehog protein, but lacking amino acidsequences at either or both of its N-terminal and C-terminal ends.Preferably such truncation mutants correspond to the amino terminal halfof a “mature” Hedgehog protein. The truncation mutants preferablycomprise at least 50-60 amino acid residues, more preferably 90-100amino acid residues, and most preferably at least 150 amino acidresidues of a Hedgehog protein, or variant thereof, while retaining atleast one activity of a Hedgehog protein. Such truncated Hedgehogpreferably have at least 63% amino acid identity with the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferablyhaving at least 75, 80, 90, 95 or 99% % amino acid identity with theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Any protein, polypeptide or truncated mutant comprised within the term“Hedgehog protein” preferably has at least one biological activity ofthe native Hedgehog proteins defined by the amino acid sequences of SEQID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the biologicalactivity of the Hedgehog proteins for use in the present invention atleast comprises one or more of: (1) the ability to bind to a Hedgehogbinding receptor Patched (Ptc), and preferably activate signallingdownstream of Ptc through the receptor Smoothened (Smo) and the Glifamily of transcriptional effectors (McMahon, 2000); (2) the ability tomaintain homeostasis of; (3) the ability to restore differentiation of;and, (4) the ability to cause gastric and/or colonic epithelial tumourcells to enter the Death program. A suitable assay for the biologicalactivity of a Hedgehog protein is to test its ability to restoredifferentiation of the HT-29 colonic cancer cell line in vitro, asdescribed in the Examples herein.

The amino acid identity between a polypeptide comprised in the term“Hedgehog” and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 may bereadily calculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Infomatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heine, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity are codified in publicly available computer programs. Preferredcomputer program methods to determine identity and similarity betweentwo sequences include, but are not limited to, the GCG program package(Devereux, J., et al., Nucleic Acids Research 12 (1):387 (1984)),BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol.Biol. 215:403-410 (1990). The BLAST X program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). The well-known Smith Waterman algorithm may also be used todetermine identity. Preferred parameters for polypeptide sequencecomparison include the following: 1) Algorithm: Needleman and Wunsch, J.Mol. Biol. 48:443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoffand Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); GapPenalty: 12; and Gap Length Penalty: 4. A program useful with theseparameters is publicly available as the “Ogap” program from GeneticsComputer Group, located in Madison, Wis. The aforementioned parametersare the default parameters for peptide comparisons (along with nopenalty for end gaps).

Operably Linked

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

Promoter

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active under mostphysiological and developmental conditions. An “inducible” promoter is apromoter that is regulated depending on physiological or developmentalconditions. A “tissue specific” promoter is only active in specifictypes of differentiated cells/tissues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery of the keyroles played by Hedgehog proteins in the regulation of homeostasis ofthe adult intestinal epithelium. We have found that Ihh is expressed inthe adult human and rodent colon, and that Ihh provides alineage-instructive signal and regulates colonic epithelialmorphogenesis in a compartmental fashion. Loss of Ihh expressionprecedes morphological change in colon tumorigenesis, i.e.carcinogenesis, and Ihh was absent in HT-29 colon carcinoma cells.Treatment of cancerous HT-29 cells with exogenous Hedgehog proteinrestored their differentiation. Ihh thus plays a pivotal role in themaintenance of colonic epithelial homeostasis in the differentiation ofthe GI tract cells and is essential for the enrolment of these GI tractcells on the Death program thus maintaining homeostasis to avoid ortreat carcinogenesis.

In addition, in gastric cancer expression of Shh was found to be lostand loss of Shh expression was found to precede morphological changes inthe parietal cells of the stomach. Expression of Shh was analysed alongthe normal human and rodent adult GI tract as well as in intestinalmetaplasia of the stomach, gastric and intestinal metaplasia of theesophagus and gastric heterotopia in Meckel's diverticulum. We foundthat Shh is specifically expressed in fundic glands as well as ingastric heterotopia in the esophagus in Meckel's diverticulum and thatShh has a unique role as a morphogen in fundic gland homeostasis.

Thus, the invention relates to Hedgehog (hh) proteins and their role inmaintaining adult intestinal homeostasis. In particular the inventionrelates to Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) expression inadult gastric and colonic, tissues respectively, whereby absence orexpression of these Hedgehog proteins (or mRNAs) leads to carcinogenesisin these tissues. While it was known in the art before the presentinvention that Hedgehog is involved in ontogenesis in various types oftissues no understanding was available regarding the role of Hedgehog inadult tissues, in particular no information was available as to the roleof Hedgehog in suppressing tumorigenesis in these tissues. We have nowfound that upregulation of Hedgehog prevents, as well as provides for atreatment of carcinogenesis in the adult gastric and colonic tissues.

The present invention thus provides for methods of, and compositions foruse in methods of diagnosis, prevention and therapy of intestinalepithelial tumorigenesis, in particular carcinogenesis of gastric andcolonic tissues, using compositions comprising Hedgehog proteins ornucleic acids coding therefor, or compositions for the detection ofthese Hedgehog molecules.

Thus in a first aspect, the present invention relates to a method oftreating an deficiency of a Hedgehog protein in the GI tract, whereinthe method comprises providing a source of Hedgehog protein to the GItract of a subject suffering from the deficiency of a Hedgehog proteinin the GI tract. The deficiency of the Hedgehog protein preferably is anacquired deficiency of the Hedgehog protein. The acquired deficiency ofthe Hedgehog protein in the GI tract may be the result of an acquiredsomatic mutation resulting in reduced expression of Hedgehog and/or asomatic activating mutation in the Wnt-β-catenin pathway. In the method,the source of Hedgehog protein may be provided to the GI tract of asubject suffering from the deficiency of a Hedgehog protein for theprophylaxis of carcinogenesis in the GI tract. Preferably the source ofHedgehog protein is provided for the prophylaxis of gastric or coloniccancer. Alternatively, the method comprises providing a source ofHedgehog protein to the GI tract of a subject suffering from theacquired deficiency of a Hedgehog protein for the treatment of a GItract carcinoma. Preferably the source of Hedgehog protein is providedfor the treatment of gastric or colonic cancer.

In a preferred embodiment the invention relates to methods of treating asubject having been diagnosed with familial adenomatous polyposis coli(FAP). The method comprises administering to a subject having beendiagnosed with FAP source of Hedgehog protein to the GI tract of thesubject. The method preferably is a method that prevents or reversestumorigenesis in the subject having been diagnosed with FAP. Preferablythe method is a method that prevents or treats GI tract tumours, inparticular, (colonic) adenomatous polyps and invasive adenocarcinomas,small intestinal adenomas and cancers, and desmoid tumors.

In these prophylactic and therapeutic methods, the source of Hedgehogprotein is administered in such an amount that functional levels ofHedgehog protein is maintained or restored in the subject's GI tract.The functional level of Hedgehog protein achieves the desiredprophylactic or therapeutic effects. Such a functional level preferablyis a level that maintains homeostasis of gastric and/or colonicepithelia, or a level that restores differentiation of tumorigenic cellsin these tissues, more preferably a level that causes such intestinalcancerous cells to enter the Death program, allowing them to finally beshed into the lumen of the GI tract. The functional level may bedetermined by any of the diagnostic methods below. During the course ofthe prophylaxis or therapy the administered amount of the source ofHedgehog protein may be adjusted based on the Hedgehog protein levelsmeasured in the relevant tissues. The norm for a functional level in agiven intestinal tissue in a given physiological condition may beestablished by determining the Hedgehog proteins levels in thecorresponding tissues under comparable conditions in healthy individualsby methods known in the art per se. However, the administered amount ofthe source of the Hedgehog protein may be therapeutic amount thateffects a supranormal level of the Hedgehog protein in GI tract. Such asupranormal level may be a factor 1.5, 2, 3, 5, 10 or higher than thenorm for a functional level of Hedgehog protein in GI tract.

In the methods of the invention, the source of Hedgehog protein may beany composition that may administered to a subject, or to organs,tissues or cells, an that is capable of effecting a functional level ofHedgehog protein in the intestinal epithelium. Thus, the source ofHedgehog protein may be a pharmaceutical composition comprising aHedgehog protein, preferably a pharmaceutical composition that issuitable for oral administration; a gene therapy vector comprising anucleotide sequence encoding a Hedgehog protein and capable ofexpression of that sequence in the relevant tissues; an (enteric)bacterium capable of colonising (parts of) the GI tract, wherein thebacterium comprises a nucleotide sequence encoding a Hedgehog protein,that confers to the bacterium the ability to secrete the Hedgehogprotein; a (stem) cell, preferably autologous, e.g. an epithelial stemcell or a peripheral mononuclear blood cell that has been transformed exvivo with a nucleotide sequence that is capable of expressing a Hedgehogprotein; a (small) molecule that (up) regulates expression of Hedgehogprotein; or a molecule that inhibits Hedgehog protein activity, such ase.g. an antibody against an Hedgehog protein. Suitable sources ofHedgehog protein are described in further detail below.

In a another aspect the invention relates to use of a Hedgehog proteinfor the manufacture of a pharmaceutical composition for the treatment ofa deficiency of a Hedgehog protein in the GI tract. The invention alsorelates to the use of a gene therapy vector comprising a nucleotidesequence encoding a Hedgehog protein, for the manufacture of apharmaceutical composition for the treatment of a deficiency of aHedgehog protein in the GI tract. The invention an enteric bacteriumcomprising a nucleotide sequence encoding a Hedgehog protein, wherebythe nucleotide sequence confers to the bacterium the ability to secretethe Hedgehog protein, for the manufacture of a pharmaceuticalcomposition for the treatment of a deficiency of a Hedgehog protein inthe GI tract. In any of these uses the treatment of the deficiency of aHedgehog protein in the GI tract may be for the prophylaxis ofcarcinogenesis in the GI tract. Preferably the treatment is for theprophylaxis of gastric or colonic cancer. Any of these uses thetreatment of the deficiency of a Hedgehog protein in the GI tract mayalso be for the treatment of a GI tract carcinoma, preferably for thetreatment of gastric or colonic cancer.

In another aspect the invention relates to methods for treating ectopicgastric tissues, such as gastric heterotopia, preferably gastricheterotopia in the esophagus in Meckel's diverticulum, whereby themethod comprises reducing the functional level and/or activity of anHedgehog protein, preferably the Shh protein, in the tissues containingthe ectopic gastric tissue. Reduction of the functional level and/oractivity of the Hedgehog protein may be achieved by administering apharmaceutical composition to the GI tract of a subject with ectopicgastric tissue, whereby the pharmaceutical composition comprises amolecule capable of reducing the functional level and/or activity, ofthe Hedgehog protein. Such a molecule may be an inhibitor of theactivity of the Hedgehog protein, such as e.g. an antibody against anHedgehog protein or against its receptor, or the molecule may be amolecule that reduces or inhibits the expression of the Hedgehogprotein, e.g. an antisense nucleic acid at least part of which iscomplementary to at least a functional part of a Hedgehog mRNA. Thecomplementary part of the antisense nucleic acid is preferably at least10, 15, 20, 30 or 40 bases long. The functional part of the HedgehogmRNA preferably comprises 5′ untranslated sequences that are necessaryfor initiation of translation or it comprises a part of the Hedgehogcoding region such that binding of the antisense nucleic acid to theHedgehog mRNA will block initiation or elongation of translation of theHedgehog mRNA. Preferably, the complementary part of antisense nucleicacid has less than 40, 25, 10% mismatches with, more preferably thecomplementary part of the antisense nucleic has no mismatches with thecorresponding sequence in the Hedgehog mRNA. Alternatively, the moleculethat reduces or inhibits the expression of the Hedgehog protein is aprotein or small molecule that interferes with expression of theHedgehog protein by binding to a functional part of the Hedgehog mRNA,Hedgehog transcription regulatory sequences, or other factors requiredfor the regulation of transcription of the Hedgehog gene.

In yet another aspect, the invention relates to methods for diagnosingthe status of a subject with respect to GI tract tumorigenesis, inparticular in gastric and colon as well as ectopic tissues. Generally inthe diagnosis for GI tract carcinogenesis, morphological markers such asthe occurrence of polyps in the colon and gastrum or the morphologicaldistinct markers such as the occurrence of gastric cells ectopicallyi.e. for instance in the esophagus are used to screen for early onset ofGI tract carcinogenesis. In certain cases of early onset tocarcinogenesis of the GI tract in particular of gastric, esophagic andcolonic tissues, no morphological distinct markers i.e. morphologicaldeviating cells can be identified. The present invention now providesfor diagnostic methods in which the level of Hedgehog protein(s) orHedgehog mRNA are determined in GI tract tissue samples, in particularsamples of gastric, esophagic and colonic tissues, whereby a finding oflower than normal levels is indicative for susceptibility for GI tractcarcinogenesis, for the progression of the sampled tissues towards thedevelopment of a carcinoma or for the presence of a carcinoma. Thenormal functional level of hedgehog protein in a given intestinal tissueunder given physiological condition may be established by determiningthe Hedgehog proteins levels in the corresponding tissues undercomparable conditions in healthy individuals by methods known in the artper se. Methods for determining Hedgehog protein levels in tissuesamples preferably use antibodies against Hedgehog proteins. Suchmethods and antibodies as well as methods for generating anti-Hedgehog(monoclonal) antibodies are provided in the Examples and are generallyknown in the art (see e.g. Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, New York). Similarly, methods and materials fordetermining the expression of Hedgehog mRNA in tissue sample areprovided in the Examples and are generally known in the art (see e.g.Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual(3^(rd) edition), Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, New York).

The invention also provides for novel marker that may be used in thediagnosis of (susceptibility for) ectopic gastric tissue such as gastricheterotopia in the esophagus in Meckel's diverticulum. Those markerscomprise next to the previously decried hh markers also Droshophila genehomologues such as human BMP2 and BMP4, of which the elevated levels inthe ectopic GI tissues provide an indication that those GI tract tissuesare susceptible to carcinogenesis. Thus the invention provide for thediagnosis of ectopic gastric tissue whereby the higher than normallevels of hh, BMP2 and/or BMP4 proteins or mRNAs in a tissue sample areindicative for the presence of ectopic gastric tissues that issusceptible for carcinogenesis. Methods for determining hh, BMP2 and/orBMP4 proteins or mRNAs and for determining the normal levels of theseproteins and mRNA's are as described above.

In a further aspect, the invention relates to a gene therapy vectorcomprising a nucleotide sequence encoding a Hedgehog protein. Nucleotidesequences encoding Hedgehog proteins, gene therapy vectors and methodsfor their construction and use are as described below.

In another aspect, the present invention relates to an enteric bacteriumcomprising a nucleotide sequence encoding a Hedgehog protein, wherebythe nucleotide sequence confers to the bacterium the ability to secretethe Hedgehog protein. Preferred bacterial hosts are capable of survivingin a mammalian GI tract, preferably capable of colonising a mucosalsurface lining of the mammalian GI tract, and preferably, not pathogenicto the mammal in which the bacterial host is to be employed. Withrespect to the latter aspect it is to be understood that the inventioncomprises the use of hosts that may normally be pathogenic to the mammal(e.g. Listeria spp., Salmonella spp. or Campylobacter spp.) but thathave been modified such that they are no longer pathogenic or virulent.Thus, a preferred microbial host will usually be a non-pathogenicbacterium capable of colonising mucosal surfaces of the mammalian GItract, which bacterium is transformed with a nucleic acid constructdescribed herein above and below. The microbial host may contain thenucleotide sequence encoding a Hedgehog protein on an episomallyreplicating molecule, or alternatively and more preferably, integratedinto its genome. The latter has the advantage of greater geneticstability. The bacterial host contains the nucleotide sequence encodinga Hedgehog protein as part of an expression construct in which thenucleotide sequence is operably linked to a promoter capable ofregulating transcription of the nucleotide sequence. The promoterpreferably is active in the host under the conditions that prevail whenthe bacterium is present in the GI tract of a mammal, more preferablyunder the conditions that prevail when the host is adhered to themucosal surfaces of the mammalian GI tract. These may be constitutivepromoters but particularly suitable promoters for this purpose arepromoters from Gram-positive bacteria that are regulated by cysteineattenuation, such as the promoter of the mapA operon of Lactococcusreuteri (NCBI accession number CAC05301). Particularly preferred is abacterial host in which the expression construct with nucleotidesequence encoding a Hedgehog protein is integrated into the bacterialgenome by means of gene replacement whereby preferably the replacedbacterial gene is a gene that is essential for growth of the bacteriumin the environment, e.g. a gene that is required for growth on mineralmedium. A preferred bacterial gene that may be used for gene replacementwith the Hedgehog expression construct is the thyA gene, which isessential for bacterial growth in the absence of exogenous thymidine(see e.g. Steidler et al., 2000, Science 289: 1352-1355). A preferredhost is a bacterium that belongs to a genus selected from the groupconsisting of Lactobacillus, Lactococcus, Leuconostoc, Streptococcus,Bifidobacterium, Bacteroides, Eubacterium, Clostridiuni, Fusobacterium,Propioizibacterium, Enterococcus, Staphylococcus, Peptostreptococcus,and Escherichia. A further preferred host is a bacterium that is aLactobacillus or Bifidobacterium species selected from the groupconsisting of L. reuteri, L. fermentum, L. acidophilus, L. crispatus, L.gasseri, L. johnsonii, L. plantarum, L. paracasei, L. murinus, L.jensenii, L. salivarius, L. minutis, L. brevis, L. gallinarum, L.amylovorus, B. bifidum, B. longum, B. infantis, B. breve, B.adolescente, B. animalis, B. gallinarum, B. magnunm, and B.therinophilum.

Pharmaceutical Compositions

In some methods, Hedgehog protein purified from mammalian, insect ormicrobial cell cultures, from milk of transgenic mammals or other sourceis administered in purified form together with a pharmaceutical carrieras a pharmaceutical composition. Methods of producing pharmaceuticalcompositions comprising Hedgehog proteins are described in are describedin U.S. Pat. Nos. 5,789,543 and 6,207,718. The preferred form depends onthe intended mode of administration and therapeutic application. Thepharmaceutical carrier can be any compatible, nontoxic substancesuitable to deliver the polypeptides to the patient. Sterile water,alcohol, fats, waxes, and inert solids may be used as the carrier.Pharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

The concentration of the Hedgehog protein in the pharmaceuticalcomposition can vary widely, i.e., from less than about 0.1% by weight,usually being at least about 1% by weight to as much as 20% by weight ormore.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable colour, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain colouringand flavouring to increase patient acceptance.

Hedgehog protein is preferably administered parentally. Hedgehog proteinfor preparations for parental administration must be sterile.Sterilisation is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilisation andreconstitution. The parental route for Hedgehog protein administrationis in accord with known methods, e.g. injection or infusion byintravenous, intraperitoneal, intramuscular, intraarterial orintralesional routes. Hedgehog protein is administered continuously byinfusion or by bolus injection. A typical composition for intravenousinfusion could be made up to contain 10 to 50 ml of sterile 0.9% NaCl or5% glucose optionally supplemented with a 20% albumin solution and 1 to50 μg of the Hedgehog protein. A typical pharmaceutical composition forintramuscular injection would be made up to contain, for example, 1-10ml of sterile buffered water and 1 to 100 μg of the Hedgehog protein ofthe present invention. Methods for preparing parenterally administrablecompositions are well known in the art and described in more detail invarious sources, including, for example, Remington's PharmaceuticalScience (15th ed., Mack Publishing, Easton, Pa., 1980) (incorporated byreference in its entirety for all purposes).

The pharmaceutical compositions of the present invention are usuallyadministered orally. Intradermal, intramuscular or intravenousadministration is also possible in some circumstances. The compositionscan be administered for prophylactic treatment of individuals sufferingfrom, or susceptible to, carcinogenesis of the GI tract in an amountsufficient to prevent, delay or reduce the severity of subsequentdisease. For therapeutic applications, the pharmaceutical compositionsare administered to a patient suffering from established disease,carcinogenesis of the GI tract, in an amount sufficient to reduce theseverity of symptoms and/or prevent or arrest further development ofsymptoms. An amount adequate to accomplish this is defined as a“therapeutically-” or “prophylactically-effective dose.” Such effectivedosages will depend on the severity of the condition and on the generalstate of the patient's health.

In the present methods, Hedgehog protein is usually administered at adosage of about 1 μ/kg patient body weight or more per week to, apatient. Often dosages are greater than 10 μg/kg per week. Dosageregimes can range from 10 μ/kg per week to at least 1 mg/kg per week.Typically dosage regimes are 10 μ/kg per week, 20 μ/kg per week, 30 μ/kgper week, 40 μ/kg week, 60 μg/kg week, 80 μ/kg per week and 120 μg/kgper week. In preferred regimes 10 μ/kg, 20 μ/kg or 40 μ/kg isadministered once, twice or three times weekly. Treatment is typicallycontinued for at least 4 weeks, sometimes 24 weeks, and sometimes forthe life of the patient. Treatment is preferably administered by oralroute. Alternatively, in some conditions it may be desirable to achievehigher than normal levels, e.g. 150% of normal levels, 200% of normallevels or even 300% of normal levels.

Nucleotide Sequences Encoding Hedgehog Proteins

Although the intended use of hh proteins produced by mammalian, insect,or microbial cell culture, or transgenic mammals or alternativelyproduced in situ in bacteria endogenous i.e. common to the flora of theGI tract is usually administered to humans, the species from which theDNA segment encoding a hh sequence is obtained is not necessarily human.Due to the high percentage of homology between the hh homologues ofdifferent species, e.g. human, mouse, Drosophila, zebrafish, and rat(available at the NCBI website: www.ncbi.nlm.nih.gov, under accessionnumbers: XM_(—)050846, NM_(—)000193, XM_(—)090366, NM_(—)010544,XM_(—)082291, AF124382, and NM_(—)017221) all hh sequences can be usedas the provide for the same functionality and are fully interchangeable.The three known hh sequences, i.e. Indian, Sonic and Desert, are equallyhomologue to such an extent that any hh variant can be applied in theinvention. This notion is exemplified by treating colon cancer cellssuccessfully with Shh resulting into differentiation and riddance of thecancerous colon cells, while the natural ligand is in fact Ihh.

The hh DNA sequence was shown to encode precursor hh proteins ofconsistent amino acids. The entire genomic sequence of hh includingIndian, Desert and Sonic of a number of different species includinghuman, mouse, rat, chicken and zebrafish sequences are known and shows ahigh percentage of homology. Also can be determined whether or notintrons are found in the various hh genes, as for instance the human Shhgene of chromosome 7 comprises 2 introns. Transgenic mammals expressingallelic, cognate and induced variants of any of the prototypicalsequence described in this reference are included in the invention. Suchvariants usually show substantial sequence identity at the amino acidlevel with known hh genes. Such variants usually hybridize to a knowngene under stringent conditions or cross-react with antibodies to apolypeptide encoded by one of the known genes. Other examples of genomicand cDNA sequences are available from GenBank or the NCBI website. Tothe extent that additional cloned sequences of hh genes are required,they may be obtained from genomic or cDNA libraries (preferably human)using known hh sequences. In genomic constructs, it is not necessary toretain all intronic sequences.

For example, some intronic sequences can be removed to obtain a smallertransgene facilitating DNA manipulations and subsequent microinjection.See Archibald et al., WO 90/05188 (incorporated by reference in itsentirety for all purposes). Removal of some introns is also useful insome instances to enhance expression levels. Removal of one or moreintrons to reduce expression levels to ensure that posttranslationalmodification is substantially complete may also be desirable. It is alsopossible to delete some or all of the non-coding exons. In sometransgenes, selected nucleotides in hh encoding sequences are mutated toremove proteolytic cleavage sites. The sequence encoding a hh protein orany functional homologue thereof may be introduced in an entericbacterium as described below, or may be incorporated in a viral ornon-viral gene therapy vector as described below or introduced as nakedDNA expression construct.

Nucleotide sequences encoding Hedgehog proteins may also be defined bytheir capability to hybridise with the nucleotide sequences encoding theamino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 3, under moderate, orpreferably under stringent hybridisation conditions. Stringenthybridisation conditions are herein defined as conditions that allow anucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC or any other solution havinga comparable ionic strength, and washing at 65° C. in a solutioncomprising about 0.1 M salt, or less, preferably 0.2×SSC or any othersolution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having about 90% or more sequenceidentity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

Recombinant Techniques and Methods for Recombinant Production ofHedgehog (Poly)Peptides

Peptides and polypeptides for use in the present invention, such e.g.the Hedgehog proteins, can be prepared using recombinant techniques inwhich a nucleotide sequence encoding the polypeptide of interest isexpressed in cultured cells such as described in Ausubel et al.,“Current Protocols in Molecular Biology”, Greene Publishing andWiley-Interscience, New York (1987) and in Sambrook and Russel (2001)“Molecular Cloning: A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, New York; bothof which are incorporated herein by reference in their entirety. Alsosee, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing sitedirected mutagenesis) and Roberts et al. (1987) Nature 328:731-734 orWells, J. A., et al. (1985) Gene 34:315 (describing cassettemutagenesis). More specifically, methods for recombinant production ofHedgehog proteins are described in U.S. Pat. No. 5,789,543.

Typically, nucleic acids encoding the desired polypeptides are used inexpression vectors. The phrase “expression vector” generally refers tonucleotide sequences that are capable of affecting expression of a genein hosts compatible with such sequences. These expression vectorstypically include at least suitable promoter sequences and optionally,transcription termination signals. Additional factors necessary orhelpful in effecting expression can also be used as described herein.DNA encoding a polypeptide is incorporated into DNA constructs capableof introduction into and expression in an in vitro cell culture.Specifically, DNA constructs are suitable for replication in aprokaryotic host, such as bacteria, e.g., E. coli, or can be introducedinto a cultured mammalian, plant, insect, e.g., Sf9, yeast, fungi orother eukaryotic cell lines. Expression constructs may also be used togenerate transgenic plant or transgenic animals capable of producing theprotein of interest. Alternatively, both viral and non-viral expressionconstructs may be employed for gene therapy as outlined below.

DNA constructs prepared for introduction into a particular hosttypically include a replication system recognized by the host, theintended DNA segment encoding the desired polypeptide, andtranscriptional and translational initiation and termination regulatorysequences operably linked to the polypeptide encoding segment. A DNAsegment is “operably linked” when it is placed into a functionalrelationship with another DNA segment. For example, a promoter orenhancer is operably linked to a coding sequence if it stimulates thetranscription of the sequence. DNA for a signal sequence is operablylinked to DNA encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide. Generally, DNAsequences that are operably linked are contiguous, and, in the case of asignal sequence, both contiguous and in reading phase. However,enhancers need not be contiguous with the coding sequences whosetranscription they control. Linking is accomplished by ligation atconvenient restriction sites or at adapters or linkers inserted in lieuthereof.

The selection of an appropriate promoter sequence generally depends uponthe host cell selected for the expression of the DNA segment. Examplesof suitable promoter sequences include prokaryotic, and eukaryoticpromoters well-known in the art (see, e.g. Sambrook and Russel, 2001,supra). The transcriptional regulatory sequences typically include aheterologous enhancer or promoter that is recognized by the host. Theselection of an appropriate promoter depends upon the host, butpromoters such as the trp, lac and phage promoters, tRNA promoters andglycolytic enzyme promoters are known and available (see, e.g. Sambrookand Russel, 2001, supra). Expression vectors include the replicationsystem and transcriptional and translational regulatory sequencestogether with the insertion site for the polypeptide encoding segmentcan be employed. Examples of workable combinations of cell lines andexpression vectors are described in Sambrook and Russel (2001, supra)and in Metzger et al. (1988) Nature 334: 31-36; For example, suitableexpression vectors can be expressed in, e.g., insect cells, e.g., Sf9cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E.coli.

In vitro mutagenesis and expression of mutant proteins are describedgenerally in Ausubel et al. (1987, supra) and in Sambrook and Russel(2001, supra). Also see, Kunkel (1985, supra; describing site directedmutagenesis) and Roberts et al. (1987, supra; describing cassettemutagenesis).

Another method for preparing polypeptides is to employ an in vitrotranscription/translation system. DNA encoding a polypeptide is clonedinto an expression vector as described supra. The expression vector isthen transcribed and translated in vitro. The translation product can beused directly or first purified. Polypeptides resulting from in vitrotranslation typically do not contain the post-translation modificationspresent on polypeptides synthesized in vivo. Methods for synthesis ofpolypeptides by in vitro translation are described by, for example,Berger & Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques, Academic Press, Inc., San Diego, Calif., 1987(incorporated herein by reference in its entirety)

Gene Therapy Constructs

Viral and or non-viral vectors (or constructs) are used for transfectingthe targeted GI tract tissue in particular the gastric and or colonictissues. The term vector refers to a nucleic acid, protein, lipid orother molecule capable of transporting a nucleic acid to which it hasbeen operatively linked. Vectors may include circular double strandedDNA plasmids and viral vectors. Some vectors are capable of autonomousreplication in a host cell into which they are introduced (such asbacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (such as non-episomal mammalianvectors) may be integrated into the genome of a host upon introductioninto the host cell, and thereby may be replicated along with the hostgenome. Certain vectors may be capable of directing the expression ofgenes to which they are operatively linked. In recombinant vectors ofthe invention, the nucleotide sequences encoding a peptide may beoperatively linked to one or more regulatory sequences, selected on thebasis of the host cells to be used for expression. The terms operativelyor operably linked mean that sequences encoding the peptide are linkedto the regulatory sequence(s) in a manner that allows for expression ofthe peptide compound. The term regulatory sequence includes promoters,enhancers, polyadenylation signals and other expression controlelements. Such regulatory elements are described in for example inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Recombinant expression vectors of theinvention may be designed for expression of the HH peptide inprokaryotic or eukaryotic cells. For example, HH peptides may beexpressed in bacterial cells such as E. Coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel; Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant hh expression vector may be transcribedand translated in vivo, for example using T7 promoter regulatorysequences and T7 polymerase. Examples of vectors for expression in yeastS. cerevisiae include pYepSec1, pMFa and pYES2. Examples of Baculovirusinclude pAc series and the pVL series. Mammalian expression vectorsinclude pCDM8, often control functions are provided by viral regulatoryelements. For example commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. Vector DNA can beintroduced into the prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques, including introducing foreignnucleic acid into a host cell using calcium phosphate or calciumchloride co-precipitation, DEA-dextran mediated transfection,lipofection, electroporation, microinjection and viral mediatedtransfection of which examples can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Laboratory press (1989), and other laboratory manuals. Methodsfor introducing DNA into mammalian cells in vivo are also known, and maybe used to deliver the vector DNA of the invention to a patient for genetherapy. A nucleic acid sequence of the invention may be delivered tocells in vivo using methods such as direct injection of DNA,receptor-mediated transfection or non-viral transfection and lipid basedtransfection, all of which may involve the use a gene therapy vectors.Defective retroviruses are well characterised for use as gene therapyvectors (Review by Miller, A. D. (1990) Blood 76:271). Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples include pLJ, pZIP, pWE and pEM, which are well known to thoseskilled in the art. (See for example Patent applications WO94/26914 andWO95/02697). For use as a gene therapy vector, the genome of anadenovirus may be manipulated so that it encodes and expresses a hhpeptide of the invention, but is inactivated in terms of itsavailability to replicate in a normal lytic viral life cycle. See forexample Berkner et al (1988) Biotechniques 6:616, Rosenfeld et al.(1991) Science 252:431-434 and Rosenfeld et al (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1234 or other strains of adenovirus (e.g. Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Recombinant adenoviruses areadvantageous, as they do not require dividing cells to be effective genedelivery vehicles and can be used to infect a wide variety of celltypes, including epithelium, endothelial, hepatocytes and muscle cells.Adeno-associated virus (AAV) may be used as a gene therapy vector fordelivery of DNA for gene therapy purposes. AAV may be used to integrateDNA into non-dividing cells (see for example Flofte et al. (1992) Am. J.Respir. Cell. Mol. Bio. 7:349-356; Samulski et al. (1989) J. Virol.63:3822-3828 and Patent applications WO91/18088; WO93/09239; U.S. Pat.No. 4,797,368; U.S. Pat. No. 5,139,941 and EP 488 528. An AAV vector maybe used to introduce DNA into cells (see for example Hermonat etal.(19⁸5) Mol. Cell. Biol. 4:2072-2081. Herpes viral and or Lenti viralgene therapy may also be adapted for use in the invention. Generalmethods for gene therapy are known in the art. See for example U.S. Pat.No. 5,399,346 by Anderson et al (incorporated herein by reference).

DESCRIPTION OF THE FIGURES

FIG. 1: Ihh is expressed by terminally differentiated enterocytes

(A) In situ hybridisation using an Ihh probe on normal human colon,detection with purple AP Substrate. The terminally differentiatedenterocytes at the tips of the crypts (arrow) produce Ihh mRNA. (B,C)Immunohistocheristry on human (B) and rat (C) adult colon using theanti-Ihh antibody and DAB detection. Ihh protein is expressed by theterminally differentiated enterocytes in both species (arrows). (D)Western blot showing expression of Villin, Ihh and loading controlβ-Actin in butyrate treated HT-29 cells. Ihh expression is induced asthe HT-29 cell differentiates. Original magnification: A-C: 80×.

FIG. 2: Expression of Ptc, BMP2, BMP4, BNF3b and Engrailed-1

Immunohistochemistry on normal rat colon, using DAB as a substrate.Expression of (A) the Hh receptor Ptc is detected in the epithelialcells throughout the crypt and in several stromal cell types (arrows).(B) BMP2 is expressed by the terminally differentiated enterocytes(arrow). (C,D) Myofibroblast-like cells (arrow, C) and some epithelialcells with endocrine cell morphology (arrow, D) express BMP4. (E) Thetranscription factor HNF3β is detected at highest levels in the nucleiof the epithelial cells at the base of the crypt and (F) in some laminapropria lymphocytes. (G and H) A similar expression pattern was foundfor Engrailed-1. Original magnification: A,B: 80×; C: 200×; D: 80×; E:100×; F,G: 60×; H: 100×.

FIG. 3 : The effect of cyclopamine treatment on the expression ofputative Hh targets

(A) Western blots showing protein levels of putative Hedgehog regulatedproteins. The first seven lanes represent colonic homogenates of sevenindividual control animals whereas the seven lanes on the right arecyclopamine treated animals. The molecular weight is indicated in kDa onthe right of each blot. (B) Quantification of blots shown in (A), meanand standard error of the relative expression compared to the mean ofthe seven controls. p values (student's t-test): Ihh, P=0.08; BMP2,P=0.25; BMP4, P=0.0001; HNF3b, P=0.001; En-1, P=0.002; GATA6, P=0.005.

FIG. 4: The effect of cyclopamine treatment on proliferation anddifferentiation

(A) Western blots showing protein levels of markers of differentiationand proliferation. The first seven lanes represent colonic homogenatesof seven individual control animals whereas the seven lanes on the rightare cyclopamine treated animals. The molecular weight is indicated inkDa on the right of each blot. (B) Quantification of blots shown in (A),mean and standard error of the relative expression compared to the meanof the seven controls. P values: Villin, P=0.02; ITF, P=0.008; Cyclin D1P=0.01; PCNA, P<0.0001. (C) Graph showing the number of BrdU labelledcells per crypt in controls and cyclopamine treated animals. Student'st-test: P=0.036.

FIG. 5: Loss of Ihh precedes morphological change in the adenomacarcinoma sequence

(A-E) Immunohistochemical detection of Ihh in human resection specimensof sporadic adenomas. (A) A sporadic adenoma (asterisks) bordering apatch of histologically normal epithelium (arrows). (B) blow-up of thearea boxed with the continuous line in (A), the terminallydifferentiated histologically normal epithelial cells show intensestaining with the anti-Ihh antibody. (C) No staining in the superficialepithelial cells of the adenoma boxed with the dotted line in (A). (D)Another example of Ihh expression in normal tissue (arrow) and (E) lossof Ihh expression in the adenomatous area of a sporadic adenoma. (F-J)Immunohistochemical detection of Thh and β-catenin in a resectionspecimen of a patient with FAP. (F) Ihh stain. Loss of Ihh expression ina few morphologically normal crypts (asterisks) with a sharp transitionto normal Ihh expression in the adjacent crypts on both sides (arrows).(G) β-catenin stain in adjacent slide. The same crypts show abnormallocalisation of β-catenin (see blow-ups). (E) Blow-up of boxed area inF. A sharp transition (arrow) can be seen between the crypt with normalIhh expression and the crypt with loss of Ihh expression. (I) Loss ofmembrane staining and cytoplasmic accumulation (arrows) of β-catenin inthe area boxed with the dotted line in (G). (J) Normal strong membranestaining (arrows) in the epithelial cells from the adjacent tissue boxedwith the continuous line in (G). Original magnifications: A: 120×; B, C:800×; D, E: 300×; F, G 500×; H-J 1000×.

FIG. 6: Exogenous Hh protein restores differentiation in HT-29 cells

Western blot showing villin expression in duplicate cultures of HT-29cells grown in a monolayer in the presence or absence of recombinant Shhfor 48 hours, using two cultures with butyrate as positive control. Thegraph depicts mean and standard error of the relative villin expressionversus control cultures in two independent experiments (n=4 percondition). Data were analysed by one-way ANOVA and Tukey's post hoccorrection. Shh versus control: P<0.01; butyrate versus control: P<0.05.

FIG. 7

Shh mRNA expression along the human GI tract. Shh expression isexpressed in the fundic glandular region (B, arrow) of the adultstomach. Shh is not detected in the esophagus (A). Minimal staining isevident in the antrum of the stomach (C), duodenum (D), and at the baseof villi in the small intestine (E, arrow at base). Shh signal in colon(F) is strongest at the base of crypts (arrow).

FIG. 8

Shh protein expression along the human and murine GI tract. (A-G)Sections of the human GI tract, Immunohistochemical staining with anantibody against the Shh precursor protein. We found intense staining inthe fundic glands (B) whereas no staining was found in esophagus (A),antrum (C), Brunner's glands (D), duodenum (E), ileum (F) or colon (G).Sections of the murine GI tract (H-N) give the same results. Shown are:forestomach (H), fundic glands (I), antral glands (J), duodenum (K),jejunum (L), ileum (M), and colon (N).

FIG. 9

Shh expression is lost in intestinal metaplasia of the stomach.Immunohistochemical triple stain of Shh (blue) MUC5AC (red) and MUC2(brown). (A-C) Specimens of two different patients with intestinalmetaplasia. (A) This specimen shows normal glands with MUC5AC expressingpit cells (arrowhead, red stain), Shh expressing gland cells (arrow,blue stain) and adjacent metaplastic glands with MUC2 expressing gobletcells (asterisks, brown stain). (B) Blow-up of boxed area in A, note theMUC5AC expression by goblet cells around the pit-gland transition. (C)Another example of metaplastic gland (asterisks) amidst normal Shhexpressing glands (arrow). (D) Although most cases showed replacement ofonly the glands or both pits and glands by intestinal cells, in thispatient we observed a mix of intestinal MUC2 expressing goblet cells(brown) and gastric MUC5AC expressing pit cells (red) in the pit region.(E-G) Three types of goblet cells were observed in this study, gobletcells that express exclusively MUC2 (arrow in E), goblet cells thatco-express MUC2 and MUC5AC (arrow in F), and goblet cells that expressonly MUC5AC (arrow in G).

FIG. 10

Shh is expressed in fundic gland ectopies. (A-C) Three different casesof Meckel's diverticulum. (A) No Shh is detected in a Meckel's specimenwith purely intestinal histology. (B) Shh staining (brown precipitate,arrow) in a gastric fundic gland adjacent to a region of goblet cellcontaining intestinal histology (asterisks). (C) Double stain of thegastric mucin MUC5AC (blue, arrow) and Shh (brown, asterisks) in aMeckel's diverticulum with fundic gland histology. (D,E) Esophagealmetaplasias. (D) No Shh is detected in the intestinal metaplasia ofBarrett's mucosa. (E) A case of fundic gland metaplasia of the esophaguswith Shh expressing cells (brown precipitate, asterisks).

FIG. 11

Cyclopamine treatment disturbs enterocyte maturation. (A-D) H&E stain ofdistal colon from control (a,c) and cyclopamine treated (b,d) animals.(C) blow-up of boxed area in A, arrows denote normal slender terminallydifferentiated enterocytes in controls. (D) blow-up of boxed area in B,arrows denote two enterocytes with normal appearance amid abnormalenterocytes with enlarged nuclei. (E,F) Villin immunohistochemistry. (E)Normal enterocytes show light cytoplasmic staining and strong stainingof the apical membrane (arrows). (F) In the abnormal appearingenterocytes of cyclopamine treated animals apical staining is diminishedand cytoplasmic staining enhanced (arrows). (G,H) CA IVimmunohistochemistry. (G) Strong staining of the enterocyte apicalmembrane in control animals (arrows). (H) Loss of carbonic anhydrase(CA) IV expression in the abnormal enterocytes in cyclopamine treatedanimals, arrows denote a few remaining CA IV expressing enterocytes.(I-L) ITF immunohistochemistry. (K) blow-up of boxed area in I, strongstaining of goblet cells (asterisks) and apical staining of enterocytes(arrow) in control colon. (L) blow-up of boxed area in J, strongimmunoreactivity of the abnormal enterocytes (arrows) in cyclopaaminetreated animals.

FIG. 12

The effect of cyclopamine treatment on proliferation in the adult ratcolon (A) Western blots showing protein levels of markers ofproliferation. The first seven lanes represent colonic homogenates ofseven individual control animals whereas the seven lanes on the rightare cyclopamine treated animals. (B) Quantification of blots (c) Graphshowing the number of BrdU labelled cells per crypt in controls andcyclopamine treated animals.

FIG. 13

Western blots of control HT-29 cells and HT-29 cells treated for 24hours. (B) Mean and standard error of 3 independent experiments(co=control, but=butyrate, cyc=cyclopamine). (C) Western blots of HT-29cells treated with 2.5 μg/ml recombinant Shh for the indicated periods.TABLE 1 The Antibodies used in this study. Name/ Antigen clone SourceProvenance IHC WB Ihh I-19 Goat Santa Cruz 1:50 1:500 Ptc C-20 GoatSanta Cruz 1:50 Ptc Rabbit Dr R. Töftgard 1:200 βActin I-19 Goat SantaCruz 1:2000 GATA-6 H-29 Rabbit Santa Cruz 1:50 1:500 Villin C-19 GoatSanta Cruz 1:1000 BMP2 mAb 355 mAb R&D systems 1:1000 1:5000 BMP4 mAB757 mAb R&D systems 1:50 1:1000 Cyclin D1 DCS6 mAb Neomarkers 1:500 En-14G11 mAb DSHB, 1:25 1:500 Dr J. M. Jessell HNF3β 4C7 mAb DSHB, 1:101:500 Dr J. M. Jessell β-catenin 14 mAb Transduction 1:1000 laboratoriesITF Rabbit Dr D. K. Podolsky 1:500Commercial antibodies were obtained from Santa Cruz (Santa Cruz, CA),R&D systems (Minneapolis, MN), Neomarkers (Fremont, CA) and Transductionlaboratories (Lexington, KY). The antibodies developed by Dr J. M.Jessell's lab, were obtained from the Developmental Studies HybridomaBank (DSHB, Iowa City, IA). IHC = immunohistochemistry, WB = Westernblot, mAb = monoclonal antibody.

EXAMPLES Materials and Methods

A. Antibodies

Antibodies used are listed below; concentrations forimmunohistochemistry are in normal font those used for immunoblot areitalicised. An anti-BMP2 mouse monoclonal antibody (mAb) (355; 1:1000;1:2000) and an anti-BMP4 mAb (757; 1:500; 1:2000) were from R&D systems(Minneapolis, Minn.). A goat polyclonal anti-Shh (N-19) that recognisesthe Shh precursor protein (van den Brink et al., 2001) (1:200; 1:2000),a goat polyclonal anti-Ihh (I-19; 1:50; 1:500), a goat polyclonalanti-Ptc (C-20, 1:50), a Rabbit polyclonal anti-GATA6 (H-29; 1:50;1:500), a goat polyclonal anti-Villin (C-19; 1:1000) and a goatpolyclonal anti-a-actin (I-19; 1:1000) were all from Santa Cruz (SantaCruz, Calif.). An anti-HNF3b mAb (4C7; 1:10; 1:1000), and ananti-Engrailed-1 mAb, both developed by Dr J. M. Jessell's laboratory,were obtained from the Developmental Studies Hybridoma Bank (Iowa City,Iowa). An anti-PCNA mAb (1:5000) and an anti-BrdU (1:100) were fromRoche (Almere, the Netherlands). Specificity of all antibodies used inimmunohistochemistry was confirmed on immunoblot. Secondary antibodiesused were all from Dako (Glostrup, Denmark). An anti-cyclin D1 mAb(DCS6) was from Neomarkers (Fremont, Calif.). An anti-b-catenin mAb(clone 14) was from Transduction laboratories (Lexington, Ky.). A rabbitpolyclonal anti-Ptc (1:200) and a rabbit polyclonal anti-ITF were a giftof Dr R. Toftgard and Dr D. K. Podolsky respectively.

For purposes of experimentation on gastric tissues a goat polyclonalα-Shh antibody (N-19, 1:250) produced by immunising with an amino acidsequence mapping at the amino terminus of the murine Shh precursor wasobtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). We haveshown previously that this antibody specifically recognises the 49-kDaShh precursor protein (van den Brink et al., 2001). A mouse monoclonalα-H⁺/K⁺-ATPase (1:6000) was from Affinity Bioreagents (Golden, Colo.). Amouse monoclonal anti-MUC5AC (1:50, clone 45M1) was from Lab Vision(Fremont, Calif.). A mouse monoclonal anti-MUC2 (1:100, clone CCP58) wasfrom Novocastra (Newcastle upon Tyne, England).

B. Immunohistochemistry and In Situ Hybridisation

Formalin fixed paraffin embedded human biopsy and resection specimens ofuninflamed colonic mucosa, sporadic adenomas, sporadic carcinomas andadenomas from patients with FAP were obtained from the archives of thepathology department of the Academic Medical Center followinginstitutional standards for human subject research. Immunohistochemistrywas performed on 4μ sections using a three-step diaminobenzidine (DAB)detection method with antigen retrieval as described in detailpreviously (van den Brink et al., 2000). For BrdU visualisation,sections were incubated in 2N HCl at 37° C. for 60 minutes afterdeparaffinization and then washed in boric acid pH 8.5. Sections werecounterstained with Mayer's hematoxylin, except when stained for HNF3β,engrailed-1 or β-catenin to allow optimal visualisation of nuclearstaining. BrdU positive nuclei were scored as described (van den Brinket al., 2001).

In situ hybridisation on paraffin sections was performed usingdigoxigenin-labeled mRNA probes for human Shh and Ihh, a gift of Dr C.Tabin.

For purposes of experimentation on gastric tissues the methods used forstaining of a single epitope on paraffin sections have been described indetail previously (van den Brink et al., 2000). For double staining ofShh and MUC5AC sections were incubated with a mixture of the anti-Shhand anti-MUC5AC overnight. The following day sections were incubatedwith a mixture of HRP coupled rabbit anti-goat Ig (1:100, Dako) andbiotinylated rabbit anti-mouse Ig (1:250). First the HRP was detectedwith fast DAB as described (Tytgat et al, 1994) and hereafter sectionswere incubated with streptavidin β-galactosidase (strep β-gal, 1:50 inPBS, Roche, Almere, The Netherlands) for 30 min at RT. The β-gal wasdetected with 40 μg/ml X-Gal (Gibco, Breda, the Netherlands) in ironphosphate buffer (0.02% MgCl₂.6H2O, 0.099% potassium ferricyanide,0.127% potassium ferrocyanide) at 37° C. for 15 min, resulting in aturquoise color.

Simultaneous immunohistochemical detection of three different epitopeswas performed largely as described previously (van den Brink et al.,2000). For triple staining of Shh, MUC5AC and MUC2 the followingprotocol was developed. Sections were rehydrated and blocked asdescribed and incubated with the anti-MUC5AC monoclonal overnight. Thefollowing day sections were incubated with an alkaline phosphatase (AP)coupled goat anti-mouse Ig (1:20, Dako) in PBS containing 10% human ABserum for one hour. After washing in Tris Buffered Saline, AP activitywas detected using Fast Red (Dako) resulting in a red precipitate.Hereafter sections were heated to 100° C. for 5 min to remove antibodiesand enhance antigen retrieval for the anti-Shh antibody. Sections wereblocked and incubated with a mixture of monoclonal anti-Muc2 and goatpolyclonal anti-Shh overnight. On day 3 sections were incubated with amixture of rabbit anti-mouse-HRP (1:50) and rabbit anti-goat-biotin(1:200) in PBS containing 10% human AB serum for one hour. First the HRPwas detected with DAB as described above and hereafter sections wereincubated with strep β-gal for 30 min at RT. The β-gal was detected asabove.

C. Immunoblot

The distal half of the rat colon was dissected along the longitudinalaxis and one half was homogenised and processed for Western blotting asdescribed below. Murine stomach and small intestine were homogenised inlysis buffer (300 mmol/L NaCl, 30 mmol/L Tris, 2 mmol/L MgCl₂, 2 mmol/LCaCl₂, 1% Triton X-100, pH 7.4, supplemented with 1 tablet of proteaseinhibitor [Roche] per 50 ml). Protein concentration was measured usingthe Bradford method. Lysates were diluted 1:3 in protein sample buffer(125 mmol/L Tris/HCl, pH 6.8; 4% sodium dodecyl sulfate; 2%β-mercaptoethanol; 20% glycerol, 1 mg bromophenol blue), and 100-200 μofhomogenate was loaded per lane on a sodium dodecylsulfate-pol-yacrylamide gel electrophoresis gel. After proteinseparation, the proteins were blotted on to a PVDF membrane (Millipore,Bedford, Mass.). Membranes were blocked with 2% protifar (Nutricia,Zoetermeer, The Netherlands) in phosphate-buffered saline (PBS),supplemented with 0.1% Tween-20 for 1 hour at room temperature. After abrief wash in washing buffer (0.2% protifar; 0.1% Tween-20), membraneswere incubated overnight at 4° C. with antibody diluted in washingbuffer at the indicated concentration. The next day, membranes werewashed 3 times for 5 minutes each and subsequently incubated with asecondary horseradish peroxidase tHRP)-conjugated antibody in a 1:2000dilution. After enhanced chemoluminescence using Lumilight⁺ substrate(Roche, Mannheim, Germany), antibody binding was visualised using aLumi-Imager (Boehringer Mannheim, Mannheim, Germany).

D. Cyclopamine Treatment

To assess a possible role of hedgehog signaling in the life cycle ofgastric epithelial cells, mice were treated with daily injections ofcyclopamine, a potent hedgehog signaling inhibitor (Incardona et al.,1998; Taipale et al., 2000; Bitgood et al.,) that inhibits hedgehogsignaling somewhere downstream of patched and upstream of thetranscriptional effectors of the Gli family, most likely at the level ofsmoothened. The cyclopamine was a kind gift of Dr. W. Gaffield. Thestudy protocol was approved by the animal ethics review board of theUniversity of Amsterdam. Cyclopamine was administered complexed with2-hydroxypropyl-β-cyclodextrin (HBC; Sigma). A cyclopamine-HBC stocksolution was produced by suspending 1 mg cyclopamine per milliliter of45% HBC in sterile PBS and stirring for 60 minutes at 65° C. Thecyclopamine-HBC was stored at −20° C. until administration. Eight7-week-old female C57BL/6 mice were given daily intraperitonealinjections of 2 mg/kg cyclopamine-HBC for 14 days. Eight mice receivedsolvent only as a control. After 14 days, mice were given a singleintraperitoneal injection of 150 mg/kg BrdU to label cells in S phase.One hour after BrdU administration, mice were killed by cervicaldislocation. To allow optimal orientation of the gastric tissue, flatstomachs were prepared according to the method described by Lee et al.and fixed in this position with needles. The stomach was transectedalong the longitudinal axis, half of the stomach was homogenized, andgastric lysates were produced as described above. The other half of thestomach and the small intestine were then fixed in 4% paraformaldehydeand embedded in paraffin. To assess proliferation of gastrointestinalepithelial cells in the cyclopamine-treated mice, gastric and smallintestinal specimens were stained with antibodies against BrdU and PCNA.Two pictures of each section were taken at 100× magnification, andpositive nuclei were counted by investigators who were blind to thetreatment in each microscope field with the use of an image analysisprogram (EFM Software, Rotterdam, The Netherlands). In each field, 5well-oriented vertical units were counted for the PCNA stain and 10 forthe BrdU stain (BrdU staining requires more counted crypts because ofthe low amount of BrdU-labeled cells). The average numbers of positivenuclei per vertical unit were compared between groups. To enablecomparison of the results between animals, all sections visualized theentire axis from the superficial epithelium to the muscularismucosa.

In addition to the mice described above, 8-week-old female Wistar rats(n=7) were treated with daily intraperitoneal injections of 1 mg/kg ofthe Hedgehog inhibitor cyclopamine complexed with2-hydroxypropyl-β-cyclodextrin (HBC; Sigma) as described (van den Brinket al., 2001). Control rats (n=7) received solvent only. After 14 days,rats were given a single intraperitoneal injection of 150 mg/kg BrdU onehour before being killed.

E. Cell Culture

HT 29 colon cancer cell lines were cultured according to routineprocedures in the presence of 10% fetal calf serum (GIBCO). RecombinantShh (R&D systems) was used at the indicated concentrations; Butyrate(Sigma) was used at a 5 mM concentration.

F. Tissues

The distal half of the rat colon was dissected along the longitudinalaxis and one half was homogenised and processed for Western blotting asdescribed (van den Brink et al., 2001). To study Shh expression in thehuman GI tract we used specimens from the archives of the department ofpathology of the Academic Medical Center and the Department of Pathologyof the Massachusetts General Hospital and Brigham and Women's Hospital.We examined tissue with normal histology of at least 6 differentpatients to investigate each site along the normal GI tract for Shhexpression. Other specimens included: tissue from 16 different patientswith intestinal metaplasia of the stomach, 13 resection specimens ofMeckel's diverticulum and 6 resection specimens of patients withBarrett's esophagus.

G. In Situ Hybridisation

Human Shh cDNA, a gift of Dr C. Tabin, was used to transcribe adigoxigenin-labeled (Roche, Mannheim, Germany) mRNA probe. Paraffinsections (4-6 μm) of archival human tissue were used for in situhybridisation using previously published methods (Robetrs et al., 1998)

Example 1 Colonic Tissues

1.1. Ihh is Expressed in the Adult Colon and Regulates the Expression ofBMP-4 and Transcription Factors Involved in Tissue Specific GeneExpression

We first examined Shh and Ihh mRNA expression in the histological normalhuman colon (FIG. 1 a) and both Shh and Ihh protein expression inhumans, rats and mice (FIGS. 1 b and c). In all three species theterminally differentiated absorptive enterocytes expressed Ihh. Althoughwe found low levels of Shh mRNA at the bases of the colonic crypts, wewere unable to demonstrate detectable Shh protein, whereas we readilydetected Shh protein in both adult human stomach and zebrafish endoderm,used as positive control (not shown). To see if Ihh correlated with thedifferentiation state in an in vitro model of enterocytedifferentiation, we next examined Ihh expression in butyrate-treatedHT29 cells (Zweibaum et al., 1985). Induction of Ihh protein expressioncorrelates well with expression of the differentiation marker Villin inthis model (FIG. 1 d).

To begin to understand the role of Hh signalling in the adult colon wefocused on the vertebrate homologues of four Drosophila genes with anestablished role in hindgut formation. These are Dpp (vertebratehomologies BMP2 and BMP4), Fork Head (vertebrate homologue HNF3β/FoxA2),Serpent (vertebrate GATA factors) and Engrailed (vertebrate Engrailed-1and 2). We localised the expression of these proteins byimmunohistochemistry and determined their relation to the Hh signal invivo in the rat using the Hh inhibitor cyclopamine. Cyclopamine is apotent Hh signalling inhibitor that inhibits Hh signalling at the levelof Smo (Taipale et al., 2000). Since only Ihh protein is detectable inthe adult colon, we presume that the effects of cyclopamine relateprincipally if not entirely to the inhibition of Ihh signalling.

We found that the Hh binding receptor and transcriptional target Ptc wasbroadly expressed in the epithelial cells along the crypt axis and onvarious mesenchymal cells (FIG. 2 a). Thus Ihh may directly affect awide range of target cells both within the epithelium and in themesenchyme. Hh genes are often co-expressed with BMP2 and BMP4 (Bitgoodet al., 1995; van den Brink et al., 2001) We found that the expressionof both morphogens in the adult colon was similar to that found duringembryonic and fetal development, BMP-2 is expressed by thedifferentiated enterocytes (FIG. 2 b), whereas we detected BNP-4 inmyofibroblast-like mesenchymal cells and in some epithelial cells in theproximal colon with endocrine cell morphology (FIGS. 2 c and d). Whereasno effect was found on the expression levels of BMP-2 upon cyclopaminetreatment, levels of BMP4 were markedly induced in response to Hhinhibition supporting the known interaction between the Hh and BMPsignalling pathways.

The transcription factors HNF3β and En-1 are highly expressed in theepithelial cells at the base of the crypt, but this expressiondiminishes towards the Ihh expressing cells at the intercrypt tables(FIG. 2 e). Both HNF3β and engrailed-1 are dramatically upregulated inresponse to cyclopamine treatment (FIG. 3). Of the GATA factors onlyGATA-6 has previously been found in colon cancer cells (Gao et al.,1998). In vivo, we observed GATA-6 expression in the terminallydifferentiated enterocytes at the intercrypt tables (not shown). Uponcyclopamine treatment GATA-6 was significantly downregulated (FIG. 3).Ihh signalling is not necessary for maintenance of its own expression(FIG. 3).

1.2. Cyclopamine Treatment Affects Both Differentiation andProliferation In Vivo

We sought to determine if cyclopamine interfered with colonic epithelialcell differentiation. We studied the expression of markers ofdifferentiation of the two main epithelial cell lineages of the colon,the enterocyte and the goblet cell. We observed a strong induction ofIntestinal trefoil factor (ITF/TFF3), a goblet cell lineage marker (FIG.4). In contrast, cyclopamine treatment reduced the expression of Villin,a cytoskeletal protein that is specific for microvilli and is a markerof enterocyte differentiation (Pringault et al., 1986). Inhibition of Hhsignalling therefore seems to interfere with enterocyte differentiationin vivo, and promote the differentiation of the goblet cell lineage.

Finally we used three markers of proliferation to assess the effect ofcyclopamine treatment on the precursor cell compartment. Cyclopaminetreatment increased both the expression of the cyclin PCNA and cyclin D1and the incorporation of 5-bromo-2′-deoxyuridine (BrdU, FIG. 4) showingthat, in the adult colon, Hh signalling may negatively regulateprecursor cell proliferation.

FIG. 11 further shows that cyclopamine treatment also disturbsenterocyte maturation in vivo. Treatment of rats with this inhibitorinhibited terminal differentiation of enterocytes in the colon, leadingto a pre-malignant state as evident by dramatic changes inhistoarchitecture, villin-redistribution, loss of carbonic anhydraseexpression, and induction of expression of intestinal trefoil factor inthe enterocytes. FIG. 12 shows that in accordance with the abovedescribed results on cell differentiation, cyclopamine treatmentenhanced mitogenesis as judged from the expression of the cyclin PCNAand cyclin D1 and the number of 5-bromo-2′-deoxyuridine (BrdU) labelledepithelial precursor cells (FIG. 12 c). We interpret these results asthat Hh signalling negatively regulates precursor cell proliferation inthe adult colon.

1.3. Loss of Ihh Expression is an Early Event in the Adenoma-CarcinomaSequence

Since we found that Ihh plays a role in the differentiation of theenterocyte lineage, we were interested to examine Ihh expression in theadenoma to carcinoma sequence of colon carcinogenesis (FIG. 5). Inresection specimens of histological normal tissue (n=3) we found Ihhstaining of all the terminally differentiated enterocytes. Intubulovillous adenomatous polyps (n=8), Ihh staining was completelylost. In contrast, we found normal Ihh staining in the morphologicallynormal tissue present in 5 of these specimens. Of the 9 adenocarcinomasstudied 6 were localised within an adenomatous polyp and 7 containednormal tissues. Ihh staining was detected in the normal epithelium butwas lost in 8 out of 9 carcinomas and in all of the adenomatous areas.In one poorly differentiated mucinous adenocarcinoma, small clusters ofpoorly differentiated cells strongly reacted with the Ihh antibody. Fromthese experiments it became evident that loss of Ihh expression alreadyoccurs at the polyp stage.

The very early lesions of the adenoma-carcinoma sequence were examinedin 10 resection specimens of four different patients with FAP (familialadenomatous polyposis coli). These patients develop numerous adenomasdue to a germline APC mutation, and somatic inactivation of thewild-type APC-allele. Multiple very small adenomas can be found in thesespecimens due to the frequent inactivation of both APC alleles in thosepatients.

We found that Ihh expression was clearly lost at the single cryptadenoma stage (not shown). In two different FAP patients we found areaswith complete loss of Ihh staining in the epithelium of a fewmorphologically normal adjacent crypts (FIG. 5 f). β-catenin staining ofconsecutive sections (FIG. 5 g) demonstrated loss of membrane stainingand cytoplasmic accumulation (FIG. 5 i) of β-catenin in the same area.The aberrant localisation of β-catenin corresponds to that describedpreviously in human aberrant crypt foci, the earliest morphologicallyrecognisable putative premalignant lesion (Hao et al., 2001). We foundthat although there is no clear morphological change in these cryptsyet, Wnt-β-catenin signalling is disturbed. Such crypts representtherefore the earliest (immunohistochemically) detectable stagefollowing an APC mutation, loss of Ihh occurred at this earliest stage.Since this loss appears to precede microscopically detectablemorphological change it may be an important step in the transition ofthe normal crypt into the adenoma. Furthermore, Ihh staining may be auseful tool to find these early lesions in large resection specimenssince it identifies lesions that cannot be detected based onmorphological criteria.

To study the very early lesions of the adenoma-carcinoma sequence, weexamined 10 resection specimens of four different patients with FAP(familial adenomatous polyposis coli).

1.4. Recombinant hh Restores HT-29 Colonic Cancer Cell DifferentiationIn Vitro.

From our in vivo data it appeared that Ihh expression in the terminallydifferentiated enterocytes signals in an autocrine manner. Loss of Ihhexpression was evident in the earliest recognisable stage in thesequence of events that is thought to progress to colon cancer. In vitrowe find that Ihh is not expressed in malignant colonic epithelial cells.The colon cancer cell HT-29 will only express Ihh protein afterdifferentiation with butyrate. We therefore decided to examine if HT-29cells can be differentiated with recombinant Hh protein in vitro. Weused recombinant Shh since this has a higher biological activity thanrecombinant Ihh, utilises the same receptor and induces the samebiological response as Ihh (Pathi et al., 2001; Yang et al., 1998).Forty-eight hour treatment of HT-29 cells induces Villin expression to asimilar extent as after treatment with 5 mM butyrate (FIG. 6). Thesedata show that exogenous Hh protein is sufficient to restoredifferentiation of colon carcinoma cells.

1.5. Butyrate Induced Differentiation of HT-29 Cells is Ihh Dependent

Treatment of HT-29 cultures with 2 μ/ml cyclopamine significantlyreduced butyrate mediated induction of cip-1, Villin and E-cadherin(FIGS. 13 a,b). Also we were able to induce the same proteins (FIG. 13c) with 2.5 μg/ml recombinant N-terminal mature murine Shh peptide,which is 91% identical with the corresponding Ihh peptide, binds Ptcwith the same affinity and induces the same biological response (Pathiet al., 2001). Thus Hh signalling is both necessary and sufficient forcolon cancer cell differentiation. Together these results show that: (i)Ihh is expressed by the terminal enterocytes; (ii) that inhibiting Hhsignalling in vivo impair enterocyte differentiation; (iii) loss of Ihhexpression precedes the development of epithelial dysplasia; and (iv)that application of Hh to colon cancer cells restores differentiation.Thus these data demonstrate that Ihh is an important factor in themaintenance of colonic epithelial homeostasis and indicate an importantrole for Ihh in the earliest stages carcinogenesis in the colon.

Example 2 Gastric Tissues

2.1. Shh Expression is Fundic Gland Specific

We have previously shown that Shh protein is expressed in the fundicglands of humans and rodents (van den Brink et al., 2001). We have nowexamined the length of the human GI tract for both Shh mRNA and proteinexpression. While no Shh mRNA was found in the normal squamousepithelium of the adult human esophagus, Shh mRNA was abundantlyexpressed in the fundic part of the stomach and at low levels in thecrypts of the small intestine and colon (as has been described in thehuman foetus, see ref 13) (FIG. 7). To investigate expression of Shhprotein we have used an antibody that recognises the Shh precursorprotein. In the human, mouse and rat GI tract Shh staining wasexclusively detected in the fundic glands of the stomach. By contrast,no Shh staining was observed in the esophagus or the intestine (FIG. 8).

2.2. Shh Expression is Lost in Intestinal Metaplasia of the Fundus

During development of the stomach, absence of Shh leads to intestinaltransformation of the stomach (de Santa Barbara et al.). In humans,replacement of gastric epithelium by epithelium of intestinal phenotype,or intestinal metaplasia, is commonly observed in patients with chronicgastritis. This metaplasia is an important risk factor for thedevelopment of gastric adenocarcinoma (Stemmermann, 1994). To evaluatethe possibility that alterations in Shh expression may be involved inintestinal metaplasia in humans, we studied whether Shh expression islost in intestinal metaplasia of the fundic gland region. To optimallylocalise Shh expression relative to the intestinal metaplasia in thesespecimens we used an immunohistochemical triple staining method. Thismethod visualises cytoplasmic and extracellular MUC5AC, the mucinproduced by gastric pit cells, cytoplasmic MUC2, a mucin specificallyexpressed by the goblet cells of the intestine (Tytgat et al., 1994),and Shh. This triple stain has an additional advantage that intestinalabsorptive enterocytes are readily identified in specimens withintestinal metaplasia due to the typical thin staining of the brushborder that contains intestinal alkaline phosphatase.

No overlap between MUC5AC and Shh was found since MUC5AC marks pit cellsthat migrate up from the precursor cell and Shh is exclusively expressedby the downward migrating parietal cells of the gastric glands (van denBrink et al., 2001). Using this method we found that the expression ofShh and MUC2 is mutually exclusive in all specimen investigated (n=16).This clearly demonstrates that in areas of intestinal metaplasia Shhexpression is completely lost.

Although not the primary aim of our study, we were also able to identifythree types of intestinal metaplasia on the basis of mucin expression,in the specimens that allowed good visualisation of the full gastricunit. The first type was characterised by MUC2 positive goblet cells andintestinal-type absorptive cells with an alkaline phosphatase positivebrush border (see unit marked with asterisks in FIG. 9D). In the secondtype, only the gland cells were replaced by MUC2 expressing goblet cellswhereas the pit cells where still of the MUC5AC expressing gastricphenotype (see FIGS. 9A-C). The third type consisted of cases the glandcontained MUC2 positive goblet cells whereas the pit consisted of a mixof MUC5AC expressing pit cells and MUC2 positive goblet cells (see unitmarked with arrow in FIG. 9D). Three types of goblet cells were observedin this study. Most goblet cells were found to express MUC2 exclusively(FIG. 9 ^(E)) as is the case in a normal intestinal goblet cell (Tytgatet al., 1994), however we also found more rarely that goblet cells candisplay exclusive expression of MUC5AC (FIG. 9G) or co-express MUC2 andMUC5AC (FIG. 9F). The MUC2-MUC5AC co-expressing goblet cells were foundin the region of the isthmus, whereas goblet cells that had migratedfurther from here were invariably found to express only MUC2 indicatinga transition from a mixed gastric-intestinal to a purely intestinalphenotype.

2.3. Shh is Expressed in Fundic Gland Heterotopia

To investigate if Shh is expressed in gastric heterotopia of the smallintestine, we examined human resection specimens of Meckel'sdiverticulum (n=13). Meckel's diverticulum is a common abnormality ofthe small intestine that occurs in 1-3% of the population (Turgeon etal., 1990). This remnant of the omphalomesenteric duct often containsheterotopic tissue of various endodermal derivatives. We stained allspecimens examined for both the H⁺K⁺ ATPase to identify acid producingparietal cells of the fundic gland and for Shh. All specimens thatcontained parietal cells (n=8) were also positive for Shh (FIGS. 10B,C),whereas specimens that lacked fundic glands (4 with intestinal and 1with antral mucosa) also lacked Shh staining (FIG. 10A). Thus Shh isexpressed in fundic gland heterotopia, indicating that aberrantdevelopment of intestinal epithelium into gastric epithelium with fundicglands is accompanied by Shh expression.

2.4. Shh is Expressed in Fundic Gland Metaplasia of the Esophagus

In patients with chronic acid reflux the resulting inflammation of theesophagus can lead to columnar metaplasia of the normally squamousepithelium of the esophagus, a condition called Barrett's esophagus.While these patients frequently develop intestinal metaplasia in thecolumnar lined segment, a mixture of gastric and intestinal-typeepithelium is commonly observed (Jankowsk et al., 2000). To see if Shhexpression is also induced postnatally in areas of gastric metaplasia weexamined oesophageal resection specimens of 6 patients with Barrett'sesophagus for expression of both the H⁺K⁺ ATPase and Shh. We found oneresection specimen with areas of gastric metaplasia of fundic typeglands. A complete overlap of H⁺K⁺ ATPase expression and Shh expressionwas found in this specimen (FIG. 10E), whereas all oesophageal(including the submucosal glands) and intestinal tissue in the resectionspecimens was negative for Shh (FIG. 10D). This indicates that theswitch in differentiation from squamous to gastric epithelial tissuewith fundic glands is accompanied by induction of Shh expression.

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1. A method of treating a deficiency of a Hedgehog protein in the GItract of a subject deficient in said protein and in need of suchtreatment, comprising providing to the GI tract of the subject acomposition that comprises a source of a Hedgehog protein.
 2. A methodaccording to claim 30, wherein the subject is at risk for, or suffersfrom cancer of the GI tract and said providing results in (i) preventionof said cancer development, or (ii) therapy of said cancer.
 3. A methodaccording to claim 2, wherein the cancer is is gastric or colon cancer.4. A method according to claim 2, wherein the from a cancer is a GItract carcinoma and said providing results in therapy of said carcinoma.5. A method according to claim 4, wherein the GI tract carcinoma is agastric or colon carcinoma.
 6. A method according to claim 1, whereinthe deficiency of the Hedgehog protein is an acquired deficiency.
 7. Amethod according to claim 30, wherein the subject has familialadenomatous polyposis coli (FAP).
 8. A method according to claim 7,wherein the Hedgehog protein, homologue or variant, nucleic acidexpression vector, bacterial delivery vehicle, animal cell or inducingor upregulating agent, is administered in an amount effective to preventor reverse GI tract tumorigenesis in the FAP subject.
 9. A methodaccording to claim 7, wherein the Hedgehog protein, homologue orvariant, nucleic acid expression vector, bacterial delivery vehicle,animal cell or inducing or upregulating agent, is administered in anamount effective to prevent or reverse colonic adenomatous polyps,invasive adenocarcinomas, small intestinal adenomas and cancers, anddesmoid tumors.
 10. A method according to claim 30, wherein the sourceof Hedgehog protein comprises the Hedgehog protein, homologue or variantin a pharmaceutical composition.
 11. A method according to claim 10,wherein the pharmaceutical composition is suitable for oraladministration.
 12. A method according to claim 30, wherein the sourceof the Hedgehog protein comprises said nucleic acid vector in apharmaceutical composition.
 13. A method according to claim 30, whereinthe source of Hedgehog protein comprises said enteric bacterium. 14-24.(canceled)
 25. A method for determining whether subject is at risk fordeveloping a GI tract tumor comprising measuring the level of a Hedgehogprotein or a Hedgehog mRNA in a GI tract tissue sample from the subject.26. A method according to claim 25, wherein the tissue sample is agastric, esophageal or colon tissue.
 27. A method for diagnosing (i) thesusceptibility of a subject to develop, or (ii) the presence in asubject ofi ectopic gastric tissue comprising, determining the level ofHedgehog, BMP2 or BMP4 mRNA and/or protein in a tissue sample from thesubject.
 28. A therapeutic composition comprising a nucleotide sequenceencoding a Hedgehog protein which composition is in the form of. (a) anucleic acid expression vector; or (b) an enteric bacterium comprisingthe nucleotide sequence which bacterium is capable of secreting theHedgehog protein when colonizing the GI system of a subject.
 29. Thetherapeutic composition of claim 28 which is said enteric bacterium .30. The method of claim 1 wherein said source of Hedgehog protein is:(a) a Hedgehog protein or an active homologue or variant thereof; (b) anucleic acid expression vector comprising a nucleotide sequence thatencodes said protein, homologue or variant; (c) an enteric bacteriumcapable of colonizing a part of the GI tract which is transformed withsaid encoding nucleotide sequence and expresses and secretes saidHedgehog protein, homologue or variant; (d) an animal cell thatexpresses and secretes said Hedgehog protein, homologue or variant; or(e) a molecule or agent that induces or upregulates expression ofHedgehog protein in said subject.
 31. The method of claim 30 whereinsaid Hedgehog protein is Shh or Ihh and said homologue or variant is ahomologue or variant of Shh or Ihh.
 32. A method for preventing thedevelopment or treating a disease or condition characterized by thepresence or growth of a Hedgehog protein-expressing ectopic gastrictissue in a subject, comprising providing to the subject or to the siteof said ectopic tissue an effective amount of a substance that reducesthe functional level or activity of the Hedgehog protein.
 33. A methodaccording to claim 32, wherein said substance is an antibody specificfor said Hedgehog protein or an antisense nucleic acid at least part ofwhich is complementary to at least a functional part of a Hedgehog mRNAthat encodes a Hedgehog protein selected from the group consisting ofhuman Desert Hedgehog protein SEQ ID NO:1, human Indian Hedgehog proteinSEQ ID NO:2, and human Sonic Hedgehog protein SEQ ID NO:3.